Kaposi's SarcomaAssociated Herpesvirus (KSHV) is a gammaherpesvirus that has been implicated in the etiology of several AIDS-associated malignancies, including Kaposi's sarcoma (KS), pleural effusion lymphoma, and Castleman's disease. The establishment, maintenance and reactivation of the latent viral genome are processes essential for virus survival and pathogenesis, and represent potential targets for antiviral therapies. During latency, the viral genomes persist as multicopy episomes packaged with cellular chromatin-associated proteins. We hypothesize that chromatin organization and chromosome dynamics are essential for KSHV genome stability during latency. This is significant because the latent reservoir of KSHV represents the major source of risk for future malignancies, and this latent reservoir is subject to complex epigenetic control of viral gene expression and DMA replication. In this application, we propose to investigate the role of chromatin organization and modification in the regulation of three critical regulatory regions of the KSHV genome. In particular, we will investigate the role of repressive chromatin at the immediate early gene ORF50 (Rta) promoter that restricts lytic cycle activation. We will also investigate the chromatin structure and cell cycle changes in nucleoproteins at the KSHV origin of plasmid replication found in the terminal repeats (TR). Finally, we have found a unique cohesin-binding site upstream of ORF73/LANA transcript. We will test the hypothesis that this unique cohesin-binding site functions in chromosome cohesion, plasmid stability, and latency transcription regulation during latency. Analysis of the specific cellular and viral factors controlling these regulatory elements of the KSHV genome should further our understanding of gammaherpesvirus latency and genome stability.